The invention relates to variants of insulin-like growth factor binding protein (IGFBP) variants, particularly to variants of IGFBP-3 which have resistance to hydrolysis, variants with altered nuclear localization sequences, and to variants with N-terminal extensions.
Growth factors are polypeptides which stimulate a wide variety of biological responses (e.g. DNA synthesis, cell division, expression of specific genes, etc.) in a defined population of target cells. A variety of growth factors have been identified, including the transforming growth factor beta family (TGF-xcex2s), epidermal growth factor and transforming growth factor alpha (the TGF-xcex1s), the platelet-derived growth factors (PDGFs), the fibroblast growth factor family (FGFs) and the insulin-like growth factor family (IGFs), which includes IGF-I and IGF-II.
IGF-I and IGF-II (the xe2x80x9cIGFsxe2x80x9d)are related in amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7.5 kilodaltons (kDa). IGF-I mediates the major effects of growth hormone, and is thus the primary mediator of growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since the treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in fetal growth. Both IGF-I and IGF-II have insulin-like activities (hence their names), and are mitogenic (stimulate cell division) for the cells in neural tissue.
Almost all IGF circulates in a non-covalently associated complex of IGF-I, insulin-like growth factor binding protein 3 (IGFBP-3) and a larger protein subunit termed the acid labile subunit (ALS), such that very little free IGF-I is detectable. The ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF-binding activity and appears to bind only to the IGF/IGFBP-3 complex (Baxter et al., J. Biol. Chem. 264(20):11843-11848, 1989), although some reports suggest that IGFBP-3 can bind to rat ALS in the absence of IGF (Lee et al., Endocrinology 136:4982-4989, 1995). The ternary complex of IGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa and has a substantially increased half-life in circulation when compared to binary IGF/IGFBP-3 complex or IGF alone (Adams et al., Prog. Growth Factor Res. 6(2-4):347-356; presented October 1995, published 1996). This ternary complex is thought to act xe2x80x9cas a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes in the concentration of free IGFxe2x80x9d (Blum et al. (1991), xe2x80x9cPlasma IGFBP-3 Levels as Clinical Indicatorsxe2x80x9d in Modern Concepts of Insulin-Like Growth Factors, pp. 381-393, E. M. Spencer, ed., Elsevier, New York). While there is essentially no excess (unbound) IGFBP-3 in circulation, a substantial excess of free ALS does exist (Baxter, J. Clin. Endocrinol. Metab. 67:265-272, 1988).
It should be noted that, while IGFBP-3 is the most abundant of the IGF binding proteins (xe2x80x9cIGFBPsxe2x80x9d), at least five other distinct IGFBPs have been identified in various tissues and body fluids. Although these proteins bind IGFs, they originate from separate genes and have distinct amino acid sequences. Unlike IGFBP-3, other circulating IGFBPs are not saturated with IGFs. IGFBP-3 is the only IGFBP which can form the 150 kDa ternary complex with IGF and ALS. The IGF and ALS binding domains of IGFBP-3 are thought to be in the N-terminal portion of the protein, as N-terminal fragments of the protein isolated from serum retain these binding activities. However, some of the other IGFBPs have also been suggested for use in combination with IGF-I as therapeutics.
The use of IGF/IGFBP-3 complex has been suggested for the treatment of a wide variety of disorders (see, for example, U.S. Pat. Nos. 5,187,151, 5,527,776, 5,407,913, 5,643,867, 5,681,818 and 5,723,441, as well as International Patent Applications Nos. WO 95/03817, WO 95/13823, and WO 96/02565. IGF-I/IGFBP-3 complex is also under development by Celtrix Pharmaceuticals, Inc., as a treatment for several indications, including recovery from burns and recovery from hip fracture surgery.
A prerequisite for the use of IGFBP-3, or any protein, as a component of a therapeutic is that the protein be stable under storage conditions. Many proteins are susceptible to non-enzymatic hydrolysis at sites in the protein which contain a xe2x80x9cAsp-Proxe2x80x9d sequence, although not all proteins which contain Asp-Pro are susceptible, nor are all Asp-Pro sites within a protein susceptible to hydrolysis (see for example, Shahrokh et al., 1994, Pharm. Res. 11(7):936-944). It appears that the susceptibility to hydrolysis of any given Asp-Pro sequence is dependent on the local environment of that site in the folded protein. If the Asp-Pro is not present at a site which is accessible to solvent and is not constrained against isomerization, then the site may be susceptible to hydrolysis. It will be apparent that the susceptibility to hydrolysis of Asp-Pro sequences in proteins without known three-dimensional structures cannot be predicted. IGFBP-3 has not been reported to be susceptible to non-enzymatic hydrolysis, and the three-dimensional structure of IGFBP-3 is not known, so susceptibility cannot be predicted.
Several reports have suggested that IGFBP-3 has its own cellular receptor, separate from the IGF receptors, and has its own set of bioactivities (Nickerson et al., 1997, Biochem. Biophys. Res. Comm. 237(3):690-69; Rajah et al., 1997, J. Biol. Chem. 272(18):12181-12188; Angelloz-Nicoud et al., 1996, Growth Regul. 6(3):130-136). Additionally, IGFBP-3, after internalization, has been shown to be targeted to the nucleus, and a nuclear localization signal (NLS) has been identified in the sequence of mature IGFBP-3 (Radulescu, 1994, Trends Biochem. Sci. 19(7):278; Jaques et al., 1997 Endocrinology 138(4):1767-1770).
Many therapeutic compounds, particularly xe2x80x98biotechxe2x80x99 compounds such as anti-sense oligonucleotides and protein hormones, suffer from problems relating to targeting and half-life. Anti-sense oligonucleotides, for example, generally suffer from both targeting and half-life problems Oligonucleotides which use xe2x80x98normalxe2x80x99 bases and linkages are rapidly eliminated from the circulation by nucleases present in serum. Even if the oligonucleotides are produced using variant nucleosides and/or linkages, they are still rapidly cleared from the circulation, primarily by the kidney and liver. Many protein-based therapeutics (e.g., antibodies, protein hormones and growth factors) suffer from very short circulating half-lives because the proteins are small enough to extravasate, which permits rapid clearance by the kidneys and other organs, or they are specifically cleared by the liver (e.g., antibodies). Covalent modification of proteins (e.g., pegylation) has been attempted to increase circulating half-life, but has been unsatisfactory.
Accordingly, there is a need in the art for stabilized variants of IGFBP-3.
There is also a need in the art for more efficiently delivering therapeutics, such that the therapeutics have increased half-life and bioavailability.
In one embodiment, the invention relates to variants of IGFBP-3 which are stabilized against non-enzymatic hydrolysis. The inventors have found that IGFBP-3 is susceptible to non-enzymatic hydrolysis at a Asp-Pro site in the protein. Additionally, the inventors have surprisingly found that a second site in the protein is susceptible to non-enzymatic hydrolysis. The inventors have designed IGFBP-3 variants which are resistant to non-enzymatic hydrolysis at these sites. Preferred hydrolysis-resistant IGFBP-3 variants include variants of the mature protein in which residues 116 and/or 135 are altered to amino acid residues other than aspartate.
In another embodiment, the invention relates to variants of IGFBP-3 which have altered nuclear localization signals. Preferred variants include mature IGFBP-3 in which residues 228 and/or 230 are altered.
In further embodiments, the invention relates to variants of IGFBP-3 in which an N-terminal extension is added. N-terminally extended IGFBP-3s include as the N-terminal extension, nucleotide-binding sequences, antigen-binding domains; protein hormones and growth factors. The inventors have surprisingly found that large N-terminal extensions may be added to IGFBP-3 without affecting the extended IGFBP-3""s ability to bind IGF and form a ternary complex with ALS.
In one N-terminally extended embodiment, the N-terminal extension is a nucleotide binding sequence. The extended IGFBP-3 of this embodiment can be formed into a complex with an IGF (preferably a xe2x80x9cnull IGFxe2x80x9d) and a nucleotide that is bound to the N-terminal extension. The complex thus administered results in greatly increased half-life and bioavailability of the nucleotide.
In a further N-terminally extended embodiment, the N-terminal extension is a peptide-binding sequence. The N-terminally extended IGFBP-3 of this embodiment can be formed into a complex with an IGF (preferably a xe2x80x9cnull IGFxe2x80x9d) and a peptide. The complex thus administered delivers the peptide, and results in greatly increased half-life and bioavailability of the peptide.
In another N-terminally extended embodiment, the N-terminal extension is an specific binding member. The N-terminally extended IGFBP-3 of this embodiment is formed into a complex with IGF (preferably a xe2x80x9cnull IGFxe2x80x9d) and administered to the patient. The complex binds the target of the specific binding member N-terminal extension, and has a greatly increased half-life and bioavailability compared to antibodies, soluble receptors, and other therapeutic specific binding members.
A further N-terminally extended embodiment utilizes a protein growth factor or hormone as the N-terminal extension. The N-terminally extended IGFBP-3 of this embodiment is formed into a complex with IGF (preferably a xe2x80x9cnull IGFxe2x80x9d) and administered to the patient. The N-terminal extension moiety can bind to its target receptor and has a greatly increased half-life and bioactivity conferred by being a part of the IGF/IGFBP-3/ALS complex.